Enhanced Oral Delivery of Protein Drugs Using ... - ACS Publications

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Enhanced Oral Delivery of Protein Drugs Using Zwitterion-Functionalized Nanoparticles to Overcome both the Diffusion and Absorption Barriers Wei Shan, Xi Zhu, Wei Tao, Yi Cui, Min Liu, Lei Wu, Lian Li, Yaxian Zheng, and Yuan Huang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b08183 • Publication Date (Web): 02 Sep 2016 Downloaded from http://pubs.acs.org on September 3, 2016

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Enhanced

ACS Applied Materials & Interfaces

Oral

Delivery

of

Protein

Drugs

Using

Zwitterion-Functionalized Nanoparticles to Overcome both the Diffusion and Absorption Barriers Wei Shan, † Xi Zhu, †,‡ Wei Tao,§ Yi Cui, † Min Liu, † Lei Wu, † Lian Li, † Yaxian Zheng† and Yuan Huang†,*

†

Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China

School of Pharmacy, Sichuan University, No. 17, Block 3, Southern Renmin Road, Chengdu 610041, China ‡

National Shanghai Center for New Drug Safety Evaluation and Research, Shanghai, 201203, China

§

School of life science, Tsinghua University, Beijing 100084, China

KEYWORDS. Nanoparticle, Zwitterion, Mucus, Epithelium, Oral Delivery, Insulin

ABSTRACT Oral delivery of protein drugs based on nanoparticulate delivery system requires permeation of the nanoparticles through mucus layer and subsequent absorption via epithelial cells. However, overcoming these two barriers needs very different or even contradictory surface properties of nanocarriers, which greatly limits the oral bioavailability of macromolecular drugs. Here we reported a simple zwitterions-based nanoparticle (NP) delivery platform, which showed a great potency in simultaneously overcoming both the mucus and epithelium barriers. The dense and hydrophilic coating of zwitterions endows the NPs with excellent mucus penetrating ability. Moreover, the zwitterions-based NPs also

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possessed excellent affinity with epithelial cells, which significantly improved (4.5-fold) the cellular uptake of DLPC NPs, compared to PEGylated NPs. Our results also indicated that this affinity was due to the interaction between zwitterions and cell surface transporter PEPT1. Moreover, the developed NPs loaded with insulin could induce a prominent hypoglycemic response in diabetic rats following oral administration. These results suggest that zwitterions-based NPs might provide a new perspective for oral delivery of protein therapeutics

1. INTRODUCTION Nanotechnology and nanomedicine have opened up new possibilities for oral delivery of protein-based therapeutics.1-3 Nanoparticles (NPs) can protect the protein drug from enzymatic degradation, and release it in a sustained manner.4 However, NPs do not guarantee improved absorption of their protein payloads, and this process can be greatly impeded by the absorption barrier of epithelial cells and the mucus barrier that covers the surface of intestinal epithelium.2, 5-6 Worse still, overcoming these two barriers requires very different, or even contradictory surface properties of NPs. Neutral and hydrophilic surfaces are favorable factors for mucus permeation7-9, while positive or hydrophobic surfaces are preferable for epithelium internalization.10-12 Orally administered NPs must first permeate through the mucus layer if they are to gain access to epithelial cells, and subsequently enter the systemic circulation. Several approaches have been proposed to facilitate mucus penetration.9, 13-16 One excellent example is to coat NPs with low-molecular-weight poly (ethylene glycol) (PEG).5, 7-9 Dense PEG coating creates a hydrophilic and electric neutral surface, which prevents the NPs to adhere to mucus constituents and facilitates their mobility. However, the


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densely coated PEG layers strongly inhibits cellular uptake and reduces the efficiency of binding to protein targets.17-18 To solve this dilemma, our group recently reported a platform by coating NPs with dissociable “mucus-inert” N-(2-hydroxypropyl) methacrylamide (HPMA) polymers.13, 15 Although this NP platform could successfully overcome both the two barriers using a progressive strategy, it requires complicated nano-assembly, and the delivery efficiency of the platform depends on the optimization of a to-be-investigated detaching process of HPMA polymer in mucus. Therefore, a simpler and well characterized NPs system that can overcome both the diffusion and absorption barriers are desired. Zwitterions are electric neutral compounds composed of a cation and anion group; examples include phosphorylcholine (PC), carboxybetaine (CB), and sulfobetaine (SB). These zwitterions are generally considered as biocompatible, bio-inert and extremely hydrophilic.18-20 NPs prepared with zwitterions are generally reported to be compact and resistant to protein-binding.20-21 Therefore, NPs with dense zwitterion coating would possess neutral and super hydrophilic surface, which might be a promising delivery vehicle for mucus permeation. Meanwhile, compared to the non-ionic PEGylated surface, the NPs composed of zwitterionic lipids possess a surface property that is similar to the phospholipid membrane of cells. Moreover, the dense cation and anion group on the surface of NP might be facilitating the interaction of the NP with cell membrane and the subsequent cellular uptake.18, 22-23 In the current study, we designed a zwitterion-based self-assembled NPs platform that possesses high mucus permeability and satisfactory cellular uptake efficiency. Dilauroyl phosphatidylcholine (DLPC, Figure 1A), composed of an extremely hydrophilic PC headgroup and hydrophobic dodecylic acid chains, was used in our study. DLPC could self-assemble on the surface of polylactic acid (PLA)-based NP to form a neutral and hydrophilic coating (Figure 1B). We studied the DPLC coating as a



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“mucus-inert” material that facilitated mucus permeation. We also examined the cellular uptake and transepithelial transport efficiency of this NP platform, and measured the in vivo therapeutic efficacy of insulin-load NP.

2. EXPERIMENTAL SECTION 2.1. Materials. Porcine insulin (28.3 IU/mg) was purchased from Wanbang Bio-Chemical (Jiangsu, China). Dilauroyl phosphatidylcholine (DLPC) was purchased from Echelon (Salt Lake City, USA); poly (lactic acid) (PLA) with an average molecular weight of 20 kDa with a terminal hydroxyl group was synthesized by Polymtek Biomaterial (Shenzhen, China). Dil was purchased from Invitrogen (Carlsbad, CA, USA); porcine mucin, resazurin and 4',6-diamidino-2-phenylindole (DAPI), from Sigma-Aldrich (St. Louis, MO, USA); glycylsarcosine (Gly-Sar), from TCI (Tokyo, Japan); and dimethyl sulfoxide (DMSO), from Regent Chemicals (Tianjin, China). 2.2. Cells and animals. In current studies, the human colonic adenocarcinoma cells (Caco-2) and mucus producing HT29-MTX-E12 cells (E12) were used. Cells were cultured in flasks using Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen, USA) with high glucose, 10% (v/v) fetal bovine serum (FBS; Gibco, USA), 1% (v/v) non-essential amino-acid, 1% (v/v) L-glutamine, 1% penicillin and streptomycin (100 IU/mL) (Hyclone, UT, USA). Cultures were incubated at 37°C in an atmosphere of 95% relative humidity and 5% CO2. Male Sprague-Dawley rats and BALB/C mice were obtained from Chengdu Dashuo Biological Technology (Chengdu, China), and all experiments were approved by the Institutional Animal Care and Use Committee of Sichuan University. 2.3. Preparation and characterization of NPs. NPs were prepared using a self-assembly method based


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on nanoprecipitation. To prepare DLPC NPs, 2 mg PLA and different amounts of DLPC were dissolved in 0.2 mL DMSO, and then the organic solution was added dropwise into 3 mL deionized water at 30°C with magnetic stirring (1000 rpm). To prepare F127 NPs and PVA NPs, 2 mg PLA was dissolved in 0.2 mL DMSO and the desired amount of F127 or PVA was dissolved in deionized water. Then the DMSO was added dropwise into 3 mL deionized water containing F127 or PVA. The resulting solution was dialyzed against deionized water using a membrane with a 12-kDa molecular weight cut-off at 4 °C, and the NPs were collected by centrifugation at 17000 g for 50 min. To prepare fluorescently labeled NPs, hydrophobic Dil (0.15%, w/w) was blended with PLA prior to addition to water. NP size and zeta potential were determined using a Malvern Zeta-size NanoZS90 (Malvern Instruments Ltd., U.K.). Their morphology was analyzed by transmission electron microscopy using a Tecnai G2 Spirit Bio TWIN microscope (FEI Company) operating at 80 kV. 2.4. Colloidal stability studies. To evaluate NP colloidal stability, freshly prepared particles were dispersed in buffer solutions at pH values of 2.5, 6.0, 6.8 or 7.4; in buffer solutions with ion concentrations of 10 or 40 Mm (ionic concentration of phosphate); in aqueous solution with 2% ovomucoid solution; in simulated gastric fluids (SGF) and simulated intestinal fluids (SIF). These suspensions were left standing for various times at room temperature, after which size was measured as described above. NPs stability was also evaluated using turbidimetry. In this assay, an increase in absorbance at 500 nm indicates aggregation.24 NPs were dispersed in 3.0 M NaCl in 10 mM PBS (pH 7.4) and incubated for different times at 25 or 37 °C. Aliquots (200 µl) were removed at each time point and absorbance was measured using a Varioskan Flash Multimode Reader (Thermo Fisher Scientific).



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2.5. Mucus permeation studies. In the current study, the native mucus was employed to evaluate the mucus penetrating ability of NPs, which was collected as previously described.25-26 In brief, undiluted human cervicovaginal mucus from women with normal vaginal flora was collected and stored in tubes at -40 °C. Before the experiment the mucus was equilibrated in a vibrator at 37 °C for 30 min at a speed of 100 rpm to ensure homogeneous dispersion of mucus 2.5.1. Mucin affinity analysis. For the measurement of particle-mucin aggregates, freshly prepared NPs were dispersed in porcine mucin solution at different concentrations (0.1, 1.0, or 2.0%, m/v), then vortexed at 100 rpm and incubated for 30 min at 37°C. The mixture was centrifuged at 613 g for 5min, and the precipitates were washed twice with PBS. Then precipitates were treated with 0.2 mL DMSO and fluorescence intensity was measured using a Varioskan Flash Multimode Reader.13 2.5.2. Modified fluorescence recovery after photobleaching (FRAP) analysis. FRAP experiments were carried out using an Olympus FV1000 confocal laser scanning microscopy. Dil-loaded NPs (10 µl) were blended with mucus gel (20 µl), and then the mixture was anchored on microscope slides. The photobleaching process was conducted in a predefined area at 100% laser power for 50s, and fluorescence intensity in that area was monitored over time. Image sets were visually inspected and discarded if the focal plane shifted during data collection. Relative florescence intensity in the region of interest was quantified using Image J software.27 2.5.3. Mucus diffusion analysis. The ability of NPs to permeate across a mucus layer was assessed using a Transwell® system (Corning Costar, NY, USA). Briefly, mucus gel (40 µl) was placed uniformly on the polycarbonate membrane and then covered with membrane filters (Merck Millipore, 2.0 µm). PBS (0.6 mL) was placed in the acceptor compartment. Test samples were diluted in PBS to a PLA


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concentration of 600 µg/mL. The experiment was started by carefully adding 0.2 mL of test samples to the donor compartment. Then the Transwell® was incubated at 37 °C in a shaker, and aliquots (80 µl) of test sample were removed from the acceptor chamber at each time point and replaced with an equal volume of pre-warmed PBS. Amount of permeated NP was quantitated using the Varioskan Flash Multimode Reader. The apparent permeability coefficient (Papp), expressed in cm sec-1, was calculated using the equation: =

1 × ×

Where dQ/dt is the rate of NP diffusion from donor side to acceptor side; C0, the initial concentration of NPs in the donor chamber; and A, membrane area (cm2). 2.5.4. Small intestinal distribution analysis. To evaluate the biodistribution of NPs in the small intestine, particles were orally administered in 0.1 mL to male BALB/C mice aged 5-6 weeks. Before administration, mice had been fasted overnight and given free access to water. At 90 min after administration, tissues were removed, opened longitudinally, flattened between two glass slides, and imaged using a fluorescence microscope (Olympus, BX53). Images were thresholded and percent of intestinal surface covered by NPs was quantified using Image J.27-28 2.6. Cellular uptake.E12 cells were seeded onto coverslips in 24-well plates (5×104cells/mL) and incubated for 2 days. Cells were washed twice with PBS and incubated for 2 h with Dil-loaded NPs at a PLA concentration of 500 µg/mL. Then cells were washed twice with cold PBS and stained with DAPI to label cell nuclei. Slides were coverslipped and observed using a confocal laser scanning microscope (FV1000). To quantitate cellular uptake using flow cytometry, cells were seeded into 24-well plates (7×104cells/well), incubated for 7 days, and treated for 2 h with Dil-loaded NPs to a PLA concentration



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of 500 µg/mL. Then cells were washed three times with PBS and analyzed immediately. To investigate the influence of mucus on cellular uptake, cells were seeded into 96-well plates (5×104 cells/mL). Cell density was estimated before experiment using the Alamar Blue assay. Mucus was removed with N-acetyl cysteine, then cells were incubated for 2 h with Dil-loaded NPs at a PLA concentration of 500 µg/mL. Cells were thoroughly washed to remove residual mucus and attached samples (Post-removal of mucus, result representing the NPs internalized in cells). Then cells were lysed using DMSO, and fluorescence intensity was measured using a Varioskan Flash Multimode Reader. To evaluate the amount of NPs trapped in mucus, cells were gently washed to preserve the mucus layer following incubation with test samples (Mild-post treatment, result representing the NPs internalized in cells + NPs trapped in mucus). NP uptake was expressed as fluorescence intensity relative to a value of 1 defined for F127 NPs uptake.13 The NPs trapped in mucus was calculated according to the following equation: NPs trapped % =

Mild − post treatment − Post − removal of mucus × 100 Mild − post treatment

To investigate the influence of PEPT1 on cellular uptake of NPs, the uptake study was performed with PEPT1 transporter inhibitor glycylsarcosine (Gly-Sar). Caco-2 cells, a PEPT1 expressed cell line, were pre-incubated with the inhibitor for 60 min, and then treated with test NPs for 2 h in the presence of inhibitor. The relative amount of cellular uptake was measured as described above. The amount of cellular uptake without inhibitor was used as a control. The interaction between NPs and E12 cells was also evaluated according to the process described above. 2.7. Transepithelial transport. E12 cells were seeded (5×104 cells/mL) into 24-well Transwell® plates fitted with polycarbonate membranes with 3.0-µm pore size. Monolayers were fed on both sides of the


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membrane every 2 days and cultured for 18-21 days before use. Transepithelial electrical resistance (TEER) was measured with an electrical resistance meter (Millicell ERS-2, Millipore) to monitor the integrity of the cell monolayer. Only monolayers with TEER >500 Ω•cm2 were used in experiments. At the start of the experiment, medium in the apical and basolateral chambers was replaced with pre-warmed Hank’s balanced salt solution (HBSS) and cells were equilibrated for 30 min at 37°C. Then the apical solution was replaced with200 µl test sample at a PLA concentration of 500 µg/mL in serum-free medium. After 10-h incubation at 37°C, the concentration of NPs in the acceptor chamber was measured. 2.8. Absorption In situ. Male Sprague-Dawley rats (180-220 g) were fasted overnight with free access to water, and then they were anesthetized. Midline laparotomy was performed to expose the jejunum, a 3-cm section of intestinal loop was ligated at both ends, and Dil-loaded nanoparticles (200 µl) were administered directly into the loop. After 2 h, the loops were removed and washed thoroughly with phosphate-buffered saline, then fixed with 4% paraformaldehyde for 4 h and dehydrated in 30% sucrose solution overnight at 4 °C. The tissues were frozen and cut into 10-µm slices and stained with DAPI. Slices were examined using confocal microscopy (FV1000, Olympus, USA). For quantitative analysis the loops were treated with 5 M NaOH solution after washed thoroughly with PBS. Samples were centrifuged and fluorescence intensity was measured using a Varioskan Flash Multimode Reader. 2.9. Insulin encapsulation and release. Insulin-loaded NPs were prepared by mixing 600 µg of porcine insulin, 2.0 mg of PLA and 800 µg of DLPC in 200 µl of DMSO; The mixture was subjected to nanoprecipitation in 3.0 ml of deionized water (30°C). For insulin-loaded F127 NPs and PVA NPs, 600 µg of porcine insulin and 2.0 mg PLA were dissolved in 0.2 mL DMSO and the desired amount of F127



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or PVA was dissolved in deionized water. Then the DMSO was added dropwise into 3 mL deionized water containing F127 or PVA. Insulin-loaded NPs were collected by centrifugation at 17 000 g for 50 min at 4 °C. Insulin in the supernatant and inside NPs was assayed using a reverse-phase high performance liquid chromatography (HPLC) method (Agilent 1200 series, CA, USA). Separation was achieved on a Diamosil C18 column (150 mm x 4.6 mm, 5 µm) with mobile phase of acetonitrile-water (28:72, contained 0.2M Na2SO4 and the pH was adjusted to 2.3 with phosphoric acid). The detection wavelength was set at 214 nm. The drug encapsulation efficiency (EE %) and drug loading efficiency (DL %) was calculated according to the following equation: amount of insulin in nanoparticle × 100 amount of insulin in nanoparticle + amount of insulin in supernatant amount of insulin in nanoparticle DL % = × 100 weight of nanoparticle

EE % =

To analyze insulin release, insulin-loaded NPs were added to 100-kDa dialysis units (Millipore) and immersed in SGF (without pepsin, 37 °C) at pH 2.5. After 2 h, the medium was replaced by SIF (without trypsin, 37 °C) at pH 6.8. At different time points, samples (200 µl) were withdrawn and the insulin remaining in NPs was measured.29 2.10. Protection from proteolysis and bioactivity of released insulin. To assess the ability of NPs to protect encapsulated insulin from proteolysis, insulin-loaded DLPC NPs were incubated at 37 °C in SIF for different times, and the remaining insulin was measured using high-performance liquid chromatography (Agilent 1200). As a control, free insulin was incubated with trypsin and analyzed under the same conditions.13 To evaluate the bioactivity of insulin after it had been encapsulated and then released from NPs, insulin-loaded DLPC NPs were prepared, insulin release was allowed to proceed for 2 h at 37°C, and the


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released insulin was collected. Before the experiment, Sprague-Dawley rats (180-220 g) were fasted with free access to water overnight, and then they were given the released insulin (2 IU/kg) or the same dose of free native insulin by subcutaneous injection. At the determined time point, the blood glucose level was measured using a blood glucose meter (JPS-6, Yicheng Biotech, Beijing, China) .27 2.11.

Hypoglycemic response in vivo and pharmacokinetics. Male Sprague-Dawley rats (180-220 g)

were injected with streptozotocin solution at 65 mg/kg as described.30 Rats that exhibited fasting blood glucose levels >300 mg/dL at 5 days after treatment were considered to be diabetic. Diabetic animals were fasted overnight but allowed free access to water. Then they were given free insulin or insulin-loaded F127 NPs, PVA NPs or DLPC NPs via gavage at a dose of 50 IU/kg (n=5 animals for treatment). In parallel, two groups of diabetic rates were given free insulin by subcutaneous injection (5 IU/kg) or orally empty DLPC NPs. Blood glucose level was measured using a glucose meter. Tail vein blood samples were taken at different time point, and plasma levels of insulin were measured using a porcine insulin ELISA kit (R&D System, USA). Area above the curve (AAC) of the blood glucose level and area under the curve (AUC) of plasma insulin concentration were computed. Oral pharmacological availability (PA%) and bioavailability (F%) were quantitated relative to subcutaneous injection using the following equations: AAC/012 × Dose3.5. × 100 AAC3.5. × Dose/012 AUC/012 × Dose3.5. F % = × 100 AUC3.5. × Dose/012

PA % =

2.12. Statistical analysis All data were presented as mean±SD and analyzed using SPSS 16.0 using the two-tailed Student’s t test.



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All experiments were performed in triplicate unless otherwise stated. The threshold of significance was defined as p

Enhanced Oral Delivery of Protein Drugs Using ... - ACS Publications

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